The SNAP25Δ3 mouse was generated with assistance from the Vanderbilt Transgenic Mouse and Embryonic Stem Cell Shared Resource in compliance with protocols approved by the Vanderbilt Institutional Animal Care and Use Committee. Transgenic mouse embryos were generated using the CRISPR-Cas9 system. The protospacer targeting construct was generated through a 24-mer oligo with a forward sequence of 5′-CACCGCAACAAAGATGCTGGGAAG-3′ annealed to 5′-AAACTTCCCAGCATCTTTGTTGC-3′. One microgram of px330 vector (Zhang Lab, Massachusetts Institute of Technology) was digested in a stoichiometric fashion with Bbs1 [New England Biolabs (NEB)] to a final concentration of 50 ng/μl for 1 hour at 37°C. The oligo was then ligated into the digested product with Quick Ligase (NEB) in a one-pot reaction in which oligo was added to a final concentration of 0.4 μM and ligated for 4 min at 25°C. Constructs were verified through Sanger sequencing. The single-stranded homology donor [Integrated DNA Technologies (IDT), Ultramer] was 126 bp in length and spanned the C-terminal final exon of SNAP25 with 48 bp of homology in either direction of the site of interest, along with the G204* mutation and a HindIII site 3′ of the G204* for the purposes of sequencing. The px330 vector and single-stranded homology donor were co-microinjected into the pronucleus of 587 B6D2 embryos, 447 of which were implanted into 40 B6D2 dams. A total of 32 pups were obtained, 2 of which contained the G204* mutation in germline cells as measured by PCR analysis of genomic DNA. To verify that the inserted transcript was correct, PCR products were then excised and ligated into pCR2.1TOPO, which was then subjected to Sanger sequencing using M13 and T7 primers. No changes other than the addition of the G204* and Hind III site were observed.

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